转录因子和miRNA在果蝇Toll通路免疫响应中的共调节作用研究

MicroRNAs(miRNAs) and Transcription factors (TFs) play important regulation roles in Toll signaling pathway of fruit fly. However, the co-regulatory roles mediated by TFs and miRNAs are poorly studied in Toll signaling pathway. Our previous works have shown that TF and miRNA might co-regulate MyD88 and orther genes in Toll signaling pathway of Drosophila melanogaster, implying that TFs and miRNAs could co-regulate the immune response of Toll signaling pathway. To reveal the co-regulatory roles and mechanisms mediated by TFs and miRNAs in Toll signaling pathway, in the current project, we will finish the RNA-seq sequencing of five different time points after G+bacteria stimulating Drosophila melanogaster male adult. Then, we will further construct the co-regulatory networks mediated by TFs and miRNAs in Toll signaling pathway of Drosophila melanogaster in five different time points after G+ bacterial stimulation, and compare the differences in regulation patterns of network within the five different time points, and discover the dynamic features of the co-regulatory networks mediated by TFs and miRNAs in Toll signaling pathway. Moreover, we will use functional genomics related methods to extensively study the functions of TF and miRNA co-regulation motifs with focusing on MyD88 to reveal the co-regulatory mechanisms mediated by TFs and miRNAs in Toll signaling pathway of Drosophila melanogaster during G+ bacterial infection. The completion of the project will provide valuable insights into the co-regulatory mechanisms mediated by TFs and miRNAs in fruit fly immune system, and also have important enlightenment to illuminate the molecular mechanism of the co-regulation mediated by TFs and miRNAs in Toll-like receptor immune signaling pathway of other insects and animals.

miRNA和转录因子(TF)在Toll通路中起重要调控作用，但TF和miRNA在果蝇Toll通路中的共调节作用却鲜有报道。我们发现TF和miRNA能共调节Toll通路中MyD88等基因，推测miRNA和TF共调节可能对Toll通路免疫响应起调控作用，为阐明果蝇Toll通路免疫响应中的TF和miRNA共调节作用及其机制，本项目拟对G+细菌刺激黑腹果蝇5个时间点的miRNA与mRNA转录组进行RNA-seq测序；构建G+细菌刺激下5个时间点Toll通路TF和miRNA共调节网络并与对照网络进行比较，发现TF-miRNA共调节模式的动态规律；对TF和miRNA共调节MyD88等基因进行功能研究，揭示TF和miRNA共调节果蝇Toll通路应对G+细菌感染响应的机制。项目完成对阐明TF和miRNA共调节在果蝇Toll通路免疫响应中的作用机制具有重要意义，并对动物TLR通路的深入研究提供重要的启示。

免疫响应的不足或过度激活都会对生物产生伤害甚至导致死亡。果蝇一直是研究动物先天免疫响应和人类疾病的重要模式动物。因此深入研究果蝇先天免疫稳态恢复机制对揭示果蝇自身以及人类健康都具有重要的理论和实践意义。转录因子(TF) 和miRNA是否参与果蝇免疫稳态恢复仍知之甚少。为此，本项目开展了TF和miRNA协同调节果蝇先天免疫响应机制的研究。研究内容和结果：1）通过对G+和G-细菌刺激后3h、12h和24h果蝇进行RNA-seq测序，发现在免疫响应中Toll和Imd通路各有93和67个差异表达的miRNA。2）遗传筛选和分子生物学实验证明miR-310~313家族、miR-963~964家族、miR-317和miR-958负调控果蝇Toll免疫响应, 而miR-9a和miR-981负调控果蝇Imd通路免疫响应，揭示miRNA通过靶向Toll和/或Imd通路中的基因或抗菌肽负调控果蝇免疫响应的机制。3）动态miRNA-mRNA调控网络揭示TF和miRNA协同调控多个信号通路抑制免疫过度激活，暗示它们在果蝇免疫稳态恢复中起到了重要作用。4）在网络分析的基础上进一步对转录因子dMyc调控miRNA参与免疫响应进行了研究，结果揭示dMyc能够直接激活miR-277转录进而抑制其靶基因imd和Tab2的表达负调控过度的免疫响应，致使果蝇Imd信号免疫响应恢复到一个新的稳态；特别是感染致死菌（E.cloacae）9 h后，高表达dMyc成年蝇的存活率显著高于野生型对照组，表明转录因子和miRNA在果蝇免疫响应的稳态维持中起重要调控作用。5）构建了一个Transfly数据库，该数据库收集了经RNAi敲除及ChIP-Seq结果注释的所有果蝇转录因子，以及敲除后得到的差异表达基因及其GO、KEGG、PPI结果和转录因子调控靶基因的调控网络。项目的执行过程中项目组已经保存了大量的缺陷型果蝇品系，获得了许多可能参与果蝇免疫响应的关键基因和miRNA数据，并且Transfly数据库还为方便查询转录因子调控的miRNA和基因提供了资源，这些关键数据的积累为深入揭示果蝇免疫稳态维持机制提供了直接的基础。本项目的完成不仅揭示了TF和miRNA能够协同调节果蝇免疫响应，而且也为进一步阐明维持果蝇先天免疫稳态的复杂调控机制提供了新的见解。